The use of recombinant proteins as therapeutics to combat disease is of great interest. For therapeutic use, the proteins must be produced and manufactured safely, efficiently and consistently. Furthermore, methods resulting in high protein yields and high translational fidelity are crucial to developing therapeutic proteins.
Unfortunately, errors in protein synthesis such as misincorporation of amino acids into the nascent polypeptide chain thereby generating sequence variations in the target protein can arise at any stage of gene expression. It has been estimated that error rates in typical cells are about 1 in 108 for DNA replication by E. coli, about 1 in 105 bases for transcription in E. coli and about 1 in 104 for codon translation in proteins produced in E. coli or other mammalian expression systems (Reynolds et al., Nat Rev Microbiol, 2010, 8:849-856; Kunkel, T A, J Biol Chem, 2004, 279:16895-16898; Rosenberge et al., Mol Gen Genet, 1981, 183:561-563; Blank et al., Biochemistry, 1986, 25:5920-5928; Kramer et al., RNA, 2007, 13: 87-96). In addition, error rates can be increased under stress conditions, such as during amino acid starvation (Parker et al., Proc Natl Acad Sci USA, 1978, 75:1091-1095) and in protein expression systems (Scorer et al., Nucleic Acids Res, 1991, 19:3511-3516). For example, in heterologous expression systems for overexpressing recombinant proteins, host cells undergo nutritional stress which in turn leads to increased amino acid misincorporation during translation. The undesired amino acid substitution can result in proteins with altered catalytic constants, specificity, and stability (Langridge, J, Aust J Biol Sci, 1974, 27:309-319; Nene et al., Mol Gen Genet, 1984, 194:166-172; Knowles J R, Science, 1987, 236:1252-1258; and Cupples et al., Genetics, 1988, 120:637-644). Amino acid error in therapeutic proteins can potentially induce deleterious immune responses and abnormal protein-protein interactions. Methods for removing misincorporated proteins have proven to be difficult during downstream purification schemes, especially since the error-free proteins are typically several orders of magnitude higher in concentration than the proteins containing misincorporations (Huang et al., Protein Sci, 2012, 21(5):625-632).
Misincorporation of serine residues for other amino acid residues, e.g., asparagine residues has been detected in antibodies (Shulman et al., Proc Natl Acad Sci USA, 1986, 83:7678-7682). Recombinant antibodies containing erroneous asparagine residues may exhibit altered structural and functional features, including altered sensitivity to proteolysis, reduced biological activity or increased immunogenicity (Yu et al., Analytical Chemistry, 81(22):9282-9290. Thus, there is a need for methods and compositions for producing proteins (e.g., antibodies) that are free of amino acid misincorporations. Such methods should generate high protein yields and maintain high translational fidelity. The present invention satisfies this need as well as others.